Antitumor agents and process for producing the same

ABSTRACT

Anticancer agents which contain as the active ingredient, a heme oxygenase inhibitory metalloporphyrin derivatives which are conjugated with amphipathic or water-soluble polymers (in particular, Zn-protoporphyrin (ZnPP) conjugated with polyethylene glycol). Because of being conjugated to amphipathic or water-soluble polymers, such as polyethylene glycol, the active ingredient can be administered by intravenous injection and can exert a remarkable anticancer effect owing to tumor selective delivery.

FIELD OF THE INVENTION

[0001] The present invention relates to anticancer agents with littleside effect and excellent tumor accumulation thereby exhibiting verypotent anticancer effect, and the preparation method of the same. Moreprecisely, it relates to anticancer agents containing as the activeingredient heme oxygenase inhibitory metalloporphyrin derivatives thatare conjugated with amphipathic or water-soluble polymers. And itrelates to also a preparation method of the same with high efficiency.

BACKGROUND TECHNOLOGY

[0002] The inventors of the present invention have investigatedrelationship between cancer growth or its suppression and activity ofheme oxygenase, and found that heme oxygenase is highly expressed intumor tissues. The heme oxygenase degrades heme and produces bilverdin,carbon monoxide and free iron in tumor or normal tissues.

[0003] Bilverdin is readily converted into bilirubin in the cells, andthis bilirubin is a very potent antioxidant. Thereby, bilirubin can be adefense molecule against active oxygen such as superoxide, H₂O₂, ornitric oxide etc that are generated by leukocytes of the hosts (cancerpatients). Namely, bilirubin, thus generated will nullify the toxicoxidative defense power against cancer cells or infecting microbes ofthe host. Therefore, if one blocks heme oxygenase, no bilirubin will beavailable and tumor cells will be killed by the oxidative moleculesgenerated by leukocytes as a result of innate defense state.

[0004] The inventors had tried to see the antitumor effect of zincprotoporphyrin (ZnPP), an inhibitor of heme oxygenase, administered intothe tumor feeding artery of tumor bearing rats thereby targeting theinhibitor into the tumor loci selectively, and they indeed confirmedantitumor effect in rats (K. Doi et al.: Br. J. Cancer 80, 1945-54,1999).

[0005] However, there are several problems to use ZnPP per se as anantitumor agent. First, it is almost insoluble in water per se, thus, wehad to use oily formulation to solubilize ZnPP, and such oily formulatedagent may be only injectable via the tumor-feeding artery, and this israther too elaborate and far advanced skill is required for thisprocedure compared with ordinary intravenous or subcutaneous injection.Second, native or original ZnPP has no guarantee for selectiveaccumulation of ZnPP in cancer tissues, and to exert tumor selectiveanticancer effect, whereas the drug will be widely distributed to wholebody besides tumor. Therefore, unexpected side effects are concerned.

[0006] On the contrary, the inventors are experts in tumor biology,particularly study on the vascular permeability of solid tumor tissues,and knowledgeable that macromolecular therapeutics would permeate moreselectively at the tumor tissue by virtue of unique anatomical characterand by the effect of multiple vascular permeability factors; andfurther, those macromolecules are retained in the tumor tissues for longperiod. Thus, this phenomenon was coined “enhanced permeability andretention (EPR)-effect” (Y. Matsumura, H. Maeda: Cancer Res. 47:6387-92, 1986; H. Maeda: In Advances in Enzyme Regulation (by G. Webered), Elsevier Scientific Ltd., Amsterdam, 41, 189-207, 2001).

[0007] According to the EPR-effect, drugs with molecular size largerthan 40,000 exhibit high concentration in blood plasma for prolongedtime, and several hours to days after intravenous injection; whereasintratumoral concentration will result in several fold higher moreprecisely in 24-48 hr time. This means, making the apparent drug sizegreater than 40,000, would make it possible for selective tumortargeting of such macromolecular drugs.

[0008] Meantime, various metal porphyrin derivatives having inhibitoryactivity against heme oxygenase, and improved method of theiradministrations as a whole were studied. The resulted finding is thatamphipathic or water soluble polymer conjugation to the metalprotporphyrins made it possible to yield water soluble metal porphyrinderivatives and they can be administered not only arterially but alsointravenously which has more versatile and easy clinical usage. Theyexhibited EPR-effect by polymer conjugation yielding highly efficientaccumulation in tumor, and enzyme inhibitory activity against hemeoxygenase is retained for long period. As a result, only 2 to 3 times ofinjections made it possible to suppress tumor growth completely in mice,which was a remarkable result.

[0009] Previously, metal porphyrin derivatives possessing heme oxygenaseinhibitory activity with amphipathic or water soluble polymerconjugation were never reported, nor were the method of theirpreparation before our own. Present inventors have developed the methodfor synthesis of amphipathic or water-soluble polymer conjugation ofmetalloporphyrin via amide linkage. Resultant polymer conjugatedmetalloporphyrin derivatives are novel series of compounds not reportedpreviously.

DISCLOSURE OF THE INVENTION

[0010] The present invention is anticancer agents containing as theactive ingredient a metalloporphyrin derivatives having inhibitoryactivity against heme oxygenase, especially Zn-protoporphyrin (ZnPP)conjugated with amphipathic polymers which are both water and lipidsoluble or water soluble polymers.

[0011] The present invention is also a series of novel useful compoundsfor an ingredient of anticancer agents where amphipathic orwater-soluble polymers and heme oxygenase inhibitory metalloporphyrinderivatives are conjugated via amide bonds, and preparation method ofthe compounds.

BRIEF EXPLANATIONS OF DRAWINGS

[0012]FIG. 1 shows the gel filtration chromatography of diaminoethanecoupled protoporphyrin and PEG-conjugated protoporphyrin.

[0013]FIG. 2 shows Lineweaver-Burk plot of PEG-ZnPP inhibitory profileagainst heme oxygenase.

[0014]FIG. 3 shows flow-cytometric analysis data where PEG-ZnPP treatedcultured cancer cells exhibiting more oxidant exposured profile.

[0015]FIG. 4 shows antitumor effect of PEG-ZnPP in mouse model withsolid tumor.

[0016]FIG. 5 shows the profile of body weight change during or afterintravenous administration of PEG-ZnPP.

MOST PREFERABLE EMBODIMENT FOR CARRYING OUT THE INVENTION

[0017] Amphipathic or water soluble polymers to be conjugated includepolyethylene glycol (PEG), poly propylene glycol (PPG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), various gelatins, and theirderivatives such as succinylated form, polyamino acids (eg. polymerizedaspartic acid, glutamic acid, lysine, alanine, glycine, proline,tyrosine, etc.), hydroxypropyl and other alkyl acrylate polymer,styrene-maleic acid copolymer (SMA), and their derivatives. Among thesepolymers with amphipathic and water-soluble characters, PEG and SMA aremore preferable. PEG with molecular weight of 2000-5000 is preferablyused.

[0018] SMA is copolymer of styrene and maleic acid in the alternativeorder where carboxyl group of maleic acid can be utilized to conjugatewith metalloporphyrin directly or indirectly. SMA can be per se or itsderivatives where maleic acid is partially esterified.

[0019] Metal porphyrin derivative is a complex porphyrin compound, wheremetal is chelated in stable coordination to the porphyrin ring, andprotoporphyrin is preferably used because of its easy availability amongporphyrin compounds.

[0020] Among the metals to be coordinated, iron that give no hemeoxygenase inhibitory action, mercury with poisonous nature, monovalentmetals which do not form coordinated chelation, can not be used.Although, various metals other than above such as zinc, tin, cobalt, andcupper can be used. Among them, tin and zinc complex are more preferred.However, tin is also known to be poisonous. Thus, ZnPP is the mostpreferable and its chemical structure of ZnPP is shown in Figure (B).

[0021] The anticancer agents of the present invention are anymacromolecular compounds obtained by conjugation of metal porphyrin withamphipathic or water-soluble polymers. However we found it difficult tocarry out the conjugation of the polymers to the metal porphyrindirectly, because metal porphyrin derivative is water-insoluble. Toundertake this chemical conjugation, it is preferable to conjugate thepolymers to porphyrin before the coordination of metal, then tocoordinate the metal.

[0022] The conjugation of porphyrin with the polymer can be facilitateddirectly as well as by introducing desired functional spacer group.

[0023] For example, in the case of synthesis of ZnPP, polymer can bedirectly conjugated to the two carboxyl groups in protoporphyrin, butthis direct conjugation method is not advantageous because of pooractivity of said carboxyl groups for this reaction. The inventors of thepresent invention studied effective synthesis methods of PEG conjugatedZnPP, and succeeded to synthesize ZnPP conjugated with PEG via amidebond (formula (A)).

[0024] (Where R in above formula means amphipathic or water-solublepolymer, and Me is a metal.)

[0025] The polymer conjugated ZnPP (B) may be synthesized by successivereaction as follows:

[0026] (1)Introduction of amino group to protoporphyrin IX;

[0027] (2)Conjugation of the polymer, and lastly

[0028] (3)Coordination of Zn into porphyrin ring.

[0029] For example, the scheme of synthesis of PEG conjugated ZnPP isshown diagrametrically by stepwise reaction as follows.

[0030] [Reaction (a)] Protoporphyrin IX (compd.(l)) is activated withethyl chloroformate in tetrahydrofuran (compd.(2)).

[0031] [Reaction (b)] Protoporphyrin with diamino group (compd.(3)) canbe obtained by addition of ethylene diamine.

[0032] [Reaction (c)] PEG is introduced into protoporphyrin ring byaddition of activated PEG (compd.4).

[0033] [Reaction (d)]. Lastly, PEG-ZnPP (compd.5) is obtained byaddition of zinc acetate into the reaction product of the Reaction (d).One can replace Zn for tin (Sn) and obtain PEG-Sn-PP by addition of tinacetate

[0034] Other than PEG, with amphipathic or water soluble polymers, suchas SMA can be attached to protoporphyrin similarly by condensationreaction of compd.(3) and SMA.

[0035] Heme oxygenase inhibitory methalloprotoporphyrin, that isconjugated with amphipathic or water-soluble polymers shown in formula(A) is selectively accumulated in solid tumor and exhibit excellentantitumor activity. Thus it is a novel and useful antitumor substance.The compd.(5), which is a typical example of the compound(A), whereinmetal is zinc, and R is PEG, was synthesized by the scheme shown (a) to(d). The chemical structure of the reaction product was confirmed byfollowing analysis.

[0036] Firstly, the evidence of the amino group of ethylene diamine thatwas introduced into protoporphyrin (compd.3) is confirmed by

[0037] (1)Infrared spectra with absorbance at 1641 cm-1, 1552 cm-1showed new formation of the amide bond in the compound (structure ofcompd.3)

[0038] (2)Determination of molecular weight of the compound by massspectroscopy (MS) showed 646, identical to the value calculated byformula based on the compd.3.

[0039] Then, PEG (mw about 5000) was coupled to amino group introducedinto the protoporphyrin (compd.3), and zinc is chelated. The structureof thus obtained ZnPP was identified by determination of the molecularweight and absorption spectra (UV/Vis).

[0040] Determination of the molecular weight showed mass of near 11,000Da by TOF/MS (time of flight-mass spectroscopy). And UV absorptionshowing max peak at 425, 543, and 583 indicating formula (5) isPEG-ZnPP.

[0041] The scheme of PEG-ZnPP synthesis using protoporphyrin IX asstarting material via reaction steps [a]-[d] is a novel manufacturingmethod.

[0042] Thus obtained polymer conjugated metalloporphyrin is readilywater-soluble and it may be used as injection solution eitherintravenously or arterially.

EXAMPLES

[0043] Process for preparing the PEG-ZnPP, inhibitory activity of thePEG-Znpp towards heme oxygenase and anticancer effect of PEG-ZnPP byintravenous injection according to the present invention shall beexplained in detail with the following examples. However it should beunderstood, that the present invention shall not limited to theseexamples.

[Example of Manufacturing] Synthesis of Polyethylene Glycol ConjugatedZnPP (PEG-ZnPP)

[0044] 100 mg of protoporphyrin IX was dissolved in 20 ml oftetrahydrofuran, and 2.45 ml of triethylamine was added to thissolution. This solution was kept at about 0° C. on ice, then 1.7 ml ofethyl chloroformate was added to this by dropwise under stirring, andallowed to react further for two hrs. Subsequently, triethylamine HClsalt being formed was removed by filtration, and 1.2 ml ethylene diaminewas added, and reaction was continued at room temperature for 24 hrs.The reaction mixture was then subjected to vacuum evaporation to removetetrahydrofuran, and solid material obtained was washed 7 times with50ml of distilled water yielding 60 mg of porphyrin derivative havingtwo amino groups per molecule (reaction a and b).

[0045] Five mg of compound (3) was dissolved in 25 ml of chloroform, and800 mg of succinimidoester of polyethylene glycol (Shearwater; PEG,MW5000) was added to this solution, and reacted for 24 hrs understirring at room temperature [reaction c].

[0046] PEG-conjugated protoporpyrin thus obtained was subjected to gelfiltration chromatography on Sephadex LH60 using chloroform as eluent.The result of the gel filtration chromatography showed unreactedaminated compound (3) did not exist in the preparation of PEG-conjugatedprotoporpyrin at all. It showed all aminated protoporphyrin reacted withPEG to form polymeric form of protoporphyrin. Unmodified protoporphyrin,if any, was eluted at fraction No. 20, where elution volume was similarto aminated protoporphyrin.

[0047] 40mg of zinc acetate was added to the PEG-PP solution and allowedfor two hrs at room temperature yielding PEG-conjugated zincprotoporphyrin (PEG-Zn-PP) (reaction d).

Experimental Example 1 Inhibitory Activity of PEG-ZnPP Against HemeOxidase

[0048] This was examined using purified heme oxygenase fraction derivedfrom rat spleen. It was assayed at 37° C. in the presence of hemin, thesubstrate of heme oxygenase, cofactor (NADPH, nicotine adeninedinucleotide), and cytosolic fraction containing bilirubin reductase, inwhich biliverdin formed by the oxygenase is converted to bilirubin.

[0049] Bilirubin was extracted with chloroform and quantified byabsorption at 465 nm. By the addition of either PEG-ZnPP, or unmodifiedZnPP, or no inhibitor, their effect on heme oxidase was examined, andthe Lineweaver-Burk plot of heme oxygenase activity was plotted duringthe inhibition by PEG-ZnPP. The result is shown in FIG. 2, indicatingthat PEG-ZnPP inhibits the heme oxygenase in a dose dependent manner,and inhibitory constant (Ki) was 0.13 μM. Mode of inhibition wascompetitive, and the value was equivalent to that of unmodified ZnPP.(Ki=0.12 μM)

Experimental Example 2 Effect of PEG-ZnPP on Cultured Tumor Cells

[0050] Lung adenocarcinoma cell line A549 cells were plated in plasticdish and after overnight culture, 5 μM and 10 μM of PEG-ZnPP dissolvedin distilled water were added to the culture dishes. Then, 8 hrs aftercultivation at 37° C., a reagent that quantifies oxidative stress,called dichlorodihydrofluorescein diacetyl ester (DCDHF) was added andallowing cell culture for 30 minutes. Under oxidative stress, this DCDHFwill be become oxidized and will fluoresce due to oxystress wasgenerated by formation of fluorescein in cells.

[0051] Quantification of fluorescence intensity represents the extent ofoxidative stress induced in the cells. Then, cultured cells weretrypsinized and recovered cells were subjected to the flow cytometroryanalysis and fluorescence cell population was quantified. The resultsare shown in FIG. 3, where the effect of PEG-ZnPP at 5 μM, 10 μM, iscompared with that of no drug. It is clear from these data in the FIG. 3that PEG-ZnPP brought about higher intracellular oxidative state in thedose dependent manner of PEG-ZnPP.

Experimental Example 3 Inhibition of Heme Oxygenase in Solid Tumor Modelin Mouse

[0052] In male ddY mice with mean body weight of 35 g, S 180 sarcomacells were implanted in the dorsal skin, and when solid tumor sizebecome 5 mm in cross diameter after about one week, PEG-ZnPP dissolvedin distilled water were injected via tail vein (i.v.) at 0.5 mg ZnPPequivalent per Kg body weight. The solid tumors were removed after 24hr, and heme oxygenase activity was quantified similarly as described inExample 1. Control mouse received distilled water without PEG-ZnPP. Thetumor specimens were obtained and treated similarly. As shown in Table1, PEG-ZnPP given i.v, (tail) showed significant reduction of the hemeoxygenase activity. Unmodified ZnPP could not be administered i.v.because of its difficulty in solubility. TABLE 1 Inhibition ofintratumor heme oxygenase by PEG-ZnPP given via the tail vein. Activityof heme oxygenase in tumor tissue. (n mol bilirubin/ Drug mg protein/hr)Control, none 4.17 ± 1.07 (P < 0.02) Group of PEG-ZnPP 2.30 ± 0.54administered Unmodified ZnPP Impossible to solubilize administered inwater (can not be injected)

Experiment 4 Antitumor Effect of PEG-ZnPP and Change of Body Weight inMice Bearing Solid Tumor

[0053] Similar to Experiment 3 above, sarcoma S180 of mice implantedunder the dosal skin of ddY mice, and after 10, 13 and 15 days aftertumor implantation, PEG-ZnPP at 30 n mole, 30 n mole and 50 n mole (3times only), respectively, was injected into the tail vein respectively(see also arrow marks in FIG. 4). Control mice received distilled waterinstead of PEG-ZnPP. Sizes of tumor were measured every week day asshown in FIG. 4. It is clear that PEG-ZnPP group showed remarkablesuppression of tumor growth compared with control group.

[0054] Body weight of both treated and non-treated mice were measuredsimultaneously as seen in FIG. 5. There was no remarkable body weightloss in the group treated with PEG-ZnPP.

[0055] Applicability of the Invention in the Industrial Sense

[0056] According to the present invention, metal porphyrin derivatives,which are inhibitory against heme oxygenase, can be made bothwater-soluble and lipid soluble uses and make the derivatives asintravenously injectable medicament by conjugation with amphipathic orwater-soluble polymers. This is a novel medicament having excellenttumor selective accumulation. An effective preparation method of thiscompound was also found.

[0057] The anticancer agents according to the present invention haveexcellent anticancer effect without generating any appreciable sideeffect or toxicity.

[0058] Thus the polymer conjugated anticancer agents according to thepresent invention are highly useful drug having excellent tumorselective targeting property with new and different mode of action frommany of the known low molecular weight anticancer drugs.

1. Anticancer agents which contain as the active ingredient hemeoxygenase inhibitory metalloporphyrin derivatives which are conjugatedwith amphipathic or water-soluble polymers.
 2. The anticancer agentsaccording to claim 1, wherein the heme oxygenase inhibitorymetalloporphyrin derivatives are complex compounds in which a metal atomis chelated to protoporphyrin in stable coordination.
 3. The anticanceragents according to claim 1, wherein amphipathic or water-solublepolymers are polyethylene glycol (PEG) or styrene maleic acid copolymer(SMA).
 4. The anticancer agents according to claim 1, wherein the hemeoxygenase inhibitory metalloporphyrin derivatives are conjugated withamphipathic or water-soluble polymers via amide bonds.
 5. The anticanceragents according to claims 1, wherein the metal is zinc.
 6. Hemeoxygenase inhibitory metalloporphyrin derivatives which are conjugatedwith amphipathic or water soluble polymers represented by the generalformula (A).

(Where “R” in above formula means amphipathic or water soluble polymersand “Me” is a metal).
 7. A preparation method of heme oxygenaseinhibitory metalloporphyrin derivatives, which are conjugated withamphipathic or water soluble polymers according to claim 6 whichcomprises, introducing ethylene diamine to protoporphyrin IX,introducing the polyethylene glycol chain into protoporphyrin by addingactivated polyethylene glycol, and finally adding a metal salt tointroduce the metal atom into the porphyrin ring.